The release of free fatty acids from cellular membranes has been implicated in attenuating the blood-brain barrier (BBB) permeability under pathological conditions. Recently we adopted a new in vitro system consisting of cerebromicrovascular endothelium grown on dextran microcarriers to study their properties related to the function of BBB under controlled conditions (Kempski and Spatz in press 1987). This method utilizes the fact that cell covered beads prevent accumulation of dyes like trypan blue (TB) in the microcarriers. Any increases in cellular permeability to TB results in reduced level of TB in the supernatant since TB that passes through the cellular layer binds tightly to the dextran beads. The aim of this investigation has been to use this model for evaluating the effects of free fatty acids on the permeability of separately cultured endothelial and smooth muscle cells derived from cerebral microvessels (Spatz et al. Brain Res, 191:577-582, 1980, 280:387-391, 1983). Both cell types excluded TB equally. There was no significantly different changes between the TB permeability of endothelium and smooth muscle cells exposed to either arachidonic or linoleic acid. Arachidonic acid leads to a 5-fold while linoleic acid leads to a 4- fold increase in cellular permeability to TB. On the other hand, palmitic acid caused little change but TB permeability of the smooth muscle appeared greater than that of the endothelial cells. Each of these changes could be reversed when the cells were washed after their incubation with free fatty acids but prior to the trypan blue albumin exposure. These results support the contention that free fatty acids can alter the permeability of cerebromicrovascular cellular elements and thus the function of BBB.